J. Nat. Prod. 2009, 72, 433–437
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Guanidine Alkaloids and Pictet-Spengler Adducts from Black Cohosh (Cimicifuga racemosa)† Tanja Go¨decke, David C. Lankin, Dejan Nikolic, Shao-Nong Chen, Richard B. van Breemen, Norman R. Farnsworth, and Guido F. Pauli* UIC/NIH Center for Botanical Dietary Supplements Research and Program for CollaboratiVe Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, UniVersity of Illinois at Chicago, Chicago, Illinois 60612 ReceiVed October 30, 2008
As an extension of work on the recently discovered nitrogenous metabolites from Cimicifuga/Actaea species, three new guanidine alkaloids have been isolated and characterized from C. racemosa (syn. A. racemosa) roots. Of these, cyclocimipronidine (1) and cimipronidine methyl ester (2) are congeners of cimipronidine (3), whereas dopargine (5) is a derivative of dopamine. By employing NMR- and MS-guided chemodiversity profiling of a polar serotonergic (5-HT7) fraction, the guanidine alkaloids were initially detected in a clinical extract of black cohosh and were isolated along with a congener of salsolinol 4, 5, and 3-hydroxytyrosol 3-O-glucoside (7). The structures of 1, 2, and 5 were confirmed by 1D and 2D NMR spectroscopy as well as LC-MS and HRMS spectroscopy. A plausible biosynthetic relationship may be inferred between the homoproline-analogue cimipronidines and the dopamine-derived Cimicifuga alkaloids. These strongly basic and frequently zwitterionic nitrogenous metabolites contribute considerable chemical diversity to the polar serotonergic fraction of black cohosh. Black cohosh (Cimicifuga racemosa (L.) Nutt., syn. Actaea racemosa L.), historically referred to as “black snakeroot”, is a Native American botanical, for which a monograph was included in the first U.S. Pharmacopoeia in 1820. During the 19th century, black cohosh emerged as an important treatment for a variety of female-related health conditions. Recent clinical trials have evaluated its efficacy for the alleviation of menopausal symptoms, such as hot flashes, night sweats, and various psychological complaints.1-5 In spite of the known beneficial effects of C. racemosa root extract, the bioactive compound(s) still need to be conclusively identified, especially for the development of a meaningful standardization procedure that is based on both the biological activity of the active principal(s) and the currently practiced chemical standardization protocol related to the content of certain abundant triterpenes. Competitive binding to the dopamine D2 receptor by an ethanolic (70% v/v) extract was observed6 as well as binding to the serotonin 5-HT1A, 5-HT1D, and 5-HT7 receptors by isopropanolic (40% v/v), ethanolic (75% v/v), and methanolic (100%) extracts.7 The 5-HT receptor subtypes, 5-HT1A and 5-HT7, of the hypothalamus have been shown to be associated with the generation of hot flashes,7,8 and in several clinical trials the efficacy of selective serotonin reuptake inhibitors (SSRIs) was demonstrated for the treatment of hot flashes (for reviews see refs 9-11). In addition, scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals has been reported for a methanolic root extract of C. racemosa.12 Upon initial detection by LC-MS analysis, cyclo-cimipronidine (1) was isolated and, along with cimipronidine methyl ester (2), characterized as a congener of the previously described lowmolecular weight guanidine alkaloid cimipronidine (3).13 Additionally, the presence of salsolinol (4)14 and a new compound, 5, which are both derived from dopamine, was substantiated by LC-MS analysis and point to shared in situ or biosynthetic pathways that involve Pictet-Spengler reactions.15-23 Compound 5 constitutes a new natural product for which the trivial name dopargine is proposed, in accordance with the naming of the well-studied tryptamine analogue trypargine.24,25 Dopargine (5) is the presumed Pictet-Spengler reaction product of dopamine with γ-guanidinobutyraldehyde (6). The latter is a catabolism product of arginine and may be considered a key intermediate in the possible subsequent † Dedicated to Dr. David G. I. Kingston of Virginia Polytechnic Institute and State University for his pioneering work on bioactive natural products. * Corresponding author. Tel: (312) 355-1949. Fax: (312)-355-2693. E-mail:
[email protected].
10.1021/np8006952 CCC: $40.75
biosynthetic pathways that eventually lead to guanidine derivatives. Figure 1 presents the proposed origin of 5 and 3. The presumed presence of 6 in extracts of black cohosh was detected by LC-MS experiments.
In addition to the isolated alkaloids, a glycosidic nitrogen-free derivative of dopamine, 3-hydroxytyrosol 3-O-glucoside (7),26-28 which is structurally related to the antioxidant hydroxytyrosol, was obtained from the same fraction. The present study links previous efforts to characterize constituents of the polar fractions of C. racemosa, which has recently led to the identification of the serotonergic active principle Nω-methylserotonin (8).29 This represented the first report of a derivative of a biogenic amine from black cohosh, and its concentrations in root extracts have been found to be consistent with the observed 5-HT7 activity. The polar partition of the serotonergic extract, which is also rich in phenolic compounds such as cimicifugic and caffeic acids, was also the source of the structurally diverse alkaloids reported here.
Results and Discussion Cimipronidine-Type Alkaloids. Compound 1 was obtained as a white, amorphous powder with a mass of 154.0986 amu (calcd 154.0980), consistent with the molecular formula C7H12N3O. The initial evidence for the structure of 1 came from the 13C NMR spectrum, which exhibited carbon resonances with chemical shifts and relative intensities very similar to those of 3. Three of the signals, namely, C-9 (δ 170.65), the guanidine C-6 (δ 149.83), and
2009 American Chemical Society and American Society of Pharmacognosy Published on Web 02/16/2009
434 Journal of Natural Products, 2009, Vol. 72, No. 3
Go¨decke et al.
Figure 1. Proposed asymmetric Pictet-Spengler reaction pathway leading to the biosynthesis of 5 that uses dopamine as precursor and involves the incorporation of γ-guanidinobutyraldehyde (6) via an iminium intermediate. Table 1. NMR Spectroscopic Data of 1 and 313 (400 MHz 1H), 2 (600 MHz 1H) 1 (D2O) δH, mult. (J) 2a 2b 3a 3b 4a 4b 5 6 8a 8b 9
d
3.580 m 3.459 md 2.128 md 1.953 md 2.279 md 1.624 md 3.952 md 2.831 dd (16.9, 4.2) 2.599 dd (16.9, 13.6)
313(D2O)
2 (MeOH-d4)
a
a
δH, mult. (J)
δCb 45.76
c
22.38c 30.78c 54.89c 149.83 34.80c
d
a
δH, mult. (J)
δCb c
3.405 m 3.290 md 1.959 md
48.30
2.075 md 1.880 md 4.187 md
31.82c
2.741 dd (16.5, 4.2) 2.492 dd (16.5, 9.1)
170.65 3.601 s
23.33c
56.19c 155.85 37.14c
172.90 52.17c
d
3.594 m 3.467 md 2.17 md 2.13 md 2.20 md 1.97 md 4.331 md 2.692 dd (15.1, 5.4) 2.410 dd (15.1, 8.4)
δCb 47.31c 22.61c,e 31.13c,e 56.99c 154.57 41.02c
179.83
a Referenced to the residual solvent signals at δH 4.700 for D2O and δH 3.210 for MeOH-d4. b The 13C NMR signals were referenced through HSQC and HMBC cross-peaks of the analytes in D2O solutions and to the residual solvent signal at δ 49.20 for MeOH-d4 solutions. c Signals showed cross-peaks in HSQC experiments. d Signals exhibit multiplet characteristics due to higher order virtual coupling effects; full spin analysis of these complex spin systems is beyond the scope of the present work and will be communicated separately. e Original assignments13 are reversed.
C-8 (δ 34.80), were shifted to higher field compared to the corresponding resonances in 3. This suggested a structural variation of 3, not involving the pyrrolidine ring moiety. The molecular weight obtained for the protonated 1 (m/z ) 154.0986) is 18 units lower than that of 3 (m/z ) 171.1078, C7H13N3O2),13 which suggested intramolecular lactam formation of 3, with expulsion of one molecule of H2O during a ring-closure reaction leading to 1. Further detailed analysis of the 1H NMR, COSY, and HSQC experiments was carried out (Table 1; Figures S1-S4, Supporting Information) and complemented by a 3D structural simulation of 1. The HMBC experiment was optimized for 4 Hz, which displayed most of the expected long-range coupling information (Figures S5 and S6, Supporting Information). The results of the NOESY analysis helped establish a relative configuration for 1 (Figure S7, Supporting Information). In addition to cross-peaks arising between geminal protons, NOESY correlations occurred between protons H-5, H-8a, H-4a, and H-3b and also between H-8b, H-4b, H-3a, and H-2b, respectively, indicating that these groups of protons are located on opposite sides of the molecule. By analogy to lactone-ring-opening reactions of natural products (e.g., coumarins), a sample of 1 was dissolved in NaOD at a pH >10 to monitor whether or not a similar lactam-ring-opening reaction could be observed using 1H NMR spectroscopy. The study showed that approximately 50% of 1 was converted to 3 during the first week of the experiment (Figure S26, Supporting Information) by slow chemical reaction. Upon addition of TFA (pH
